Use of xenogenized (modified) tumor cells for treatment in experimental tumor and in human neoplasia.

The need to modify tumor cells in order to render them more "immunogenic" was based on the assumption that normal, nonmodified tumor cells are non- or weakly immunogenic and as such are unable to raise an efficient protective immune response. Various methods for "xenogenization" (modification of tumor cells) were suggested: induction of new foreign antigens, treatment with either chemicals or enzymes and use of mutagens. Xenogenized tumor cells by their coupling to proteins, and use of chemicals like DTIC (5-[3,3-dimethyl- 1-triazeno]-imidazole-4-carboxamide), TZC (8-carbamoyl-3-methyl-imidazo[5, 1-d]- 1,2,3,5-tetrazin-4 [3H]-one 8-carbamoyl-3-[2-chloroethyl] imidazole [5,1 -d]- 1,2,3,5-tetrazin-4[3H]-one) and antiemetic drugs, were tested in experimental models of murine leukemia. Non-tumorigenic clones, xenogenization with DNA hypomethylating agents, aryl-triazine derivatives and DTIC were evaluated for their induction of protective immune response in murine lymphoma. Murine plasmacytoma cells were used for immunization after treatment with glutaraldehyde. Viral modifications of tumor cells were evaluated for their ability to induce a protective tumor response in model systems of rat fibrosarcoma, liver metastatic rat tumor cells, lymphoid tumor cells and hamster tumor cells. In the case of human cancer, attempts were reported to use DNP-conjugated melanoma cells, mutagenic triazine compounds, an autologous colon tumor cell bacillus Calmette-Guerin (BCG) vaccine and genetically engineered vaccines for immunization. The general conclusion drawn from experimental tumor models and for human cancer is, that although modified tumor cells were found to be partially effective in experimental models, it is still necessary to provide more data in order to determine the effective use of xenogenized human tumor cells for immunotherapy.

Rational approaches to reduce adverse reactions in man to vaccines containing tetanus and diphtheria toxoids.

Adverse reactions to routine vaccines are obstacles to the mass vaccination campaigns. Though the absolute safety of any injectable vaccine cannot be guaranteed, the adverse side effects to vaccines can be minimized by practicing existing scientific knowledge. Adverse side effects to tetanus and diphtheria toxoids have been known for many years and there have been ways to minimize these reactions. These procedures did not get wide acceptance, because the current partially purified tetanus and diphtheria vaccines meet the regulatory requirements and the manufacturers are reluctant to change the established procedures of production due to the amount of work involved in the regulatory issues under the current Good Manufacturing Practices (GMP). Due to the recent epidemic of diphtheria in the independent states of the former Soviet Union, and its potential for spread to other European Countries, vaccination campaigns with tetanus and diphtheria vaccines received a new boost with several international agencies. In this report, we review the causes for adverse reactions to tetanus and diphtheria vaccines and offer practical suggestions for minimizing these reactions. The major issues in minimizing adverse reactions to these vaccines include: (1) purifying the toxins before detoxification as the reactogenic accessory antigens get covalently bound to the toxins during detoxification; (2) either using well-tolerated adjuvants which do not elicit the production of antigenic specific IgE antibodies responsible for adverse reactions or by using non-adjuvanted highly immunogenic polymerized antigens; (3) checking the status of immunity by recently developed rapid serological methods or by the Schick skin-test for diphtheria to avoid allergic or Arthus-type reactions. These approaches are applicable to industrial scales and would result in a pure, less reactogenic and better characterized toxoids antigens which would be more suitable for combined vaccines comprising highly purified acellular pertussis components, polysaccharide-protein conjugates and other antigens.

Passive haemagglutination tests using purified antigens covalently coupled to turkey erythrocytes.

Passive haemagglutination tests have been developed by covalent coupling purified antigens to turkey red blood cells. Circulating antibodies can be assessed in 20 minutes using one drop of blood. False positive reactions are avoided by using highly purified antigens; sensitized erythrocytes are stable in the absence of freeze-drying and blood samples can be preserved on paper discs. This method, applied to the determination of circulating tetanus (T) and diphtheria (D) antibodies and titres compared to other in vivo or in vitro methods, gave good correlation. The titration of circulating antibodies can be applied in emergency care units and field trials to establish whether the individuals are adequately protected. Results of surveys by several health care centres have shown that tetanus immune coverage was insufficient in France. The decrease of both T and D immune coverage with age has been established. The antibody response of pregnant women, vaccinated with two different adsorbed T toxoids exhibiting a low and a high titre as expressed in international immunizing units (I.I.U.), was studied. No significant difference in circulating antibody titres was obtained after the first injection of either vaccine, but titres after second injection were much higher for the vaccine having a low value expressed in I.I.U. The activity of commercial and reference T vaccines can be evaluated in mice after immunization and titration of the antitoxin levels. This simple method is much easier than the official evolution of immunodeficiency in certain diseases. The passive haemagglutination test has also been used to measure anti-HBs and anti-gp 160 antibodies.

Adjuvants--a balance between toxicity and adjuvanticity.

Adjuvants have been used to augment the immune response in experimental immunology as well as in practical vaccination for more than 60 years. The chemical nature of adjuvants, their mode of action and the profile of their side effects are highly variable. Some of the side effects can be ascribed to an unintentional stimulation of different mechanisms of the immune system whereas others may reflect general adverse pharmacological reactions. The most common adjuvants for human use today are still aluminium hydroxide, aluminium phosphate and calcium phosphate although oil emulsions, products from bacteria and their synthetic derivatives as well as liposomes have also been tested or used in humans. In recent years monophosphoryl lipid A, ISCOMs with Quil-A and Syntex adjuvant formulation (SAF) containing the threonyl derivative of muramyl dipeptide have been under consideration for use as adjuvants in humans. At present the choice of adjuvants for human vaccination reflects a compromise between a requirement for adjuvanticity and an acceptable low level of side effects.

Adverse reactions after injection of adsorbed diphtheria-pertussis-tetanus (DPT) vaccine are not due only to pertussis organisms or pertussis components in the vaccine.

Reactions to adsorbed diphtheria-pertussis-tetanus (DPT) vaccine have mostly been attributed to the pertussis organisms or pertussis components in the vaccine. Nevertheless reactions may also be due to other factors such as sensitization induced by aluminium adjuvants and impurities present in crude toxoids that cannot be removed by purification of toxoids after formalinization. Aluminium compounds such as aluminium phosphate and aluminium hydroxide are the most commonly used adjuvants with vaccines for human use. Due to the increasing concern about the toxicity of aluminium, other adjuvants like calcium phosphate may be evaluated as an alternative to aluminium adjuvants. To minimize reactions after immunization with DPT vaccine due to impurities in the toxoids, the use of toxoided purified toxins is suggested.

Induction of protective antitumor immune response against a murine plasmacytoma not curable by alkylating-drug treatment.

Immunization with viable tumor cells followed by subsequent administration of glutaraldehyde-treated tumor cells induced a protective antitumor immune response in the host toward the alkylating-drug resistant RPC-5 plasmacytoma. This was proven by resistance to challenge with RPC-5 tumor cells, neutralization in Winn tests, by effectiveness of combined chemotherapy with melphalan plus immunotherapy with spleen cells from RPC-5 immunized mice and in vitro by cytotoxicity tests. The specificity of the immune response was ascertained in vivo by comparison with the response toward MOPC-315 plasmacytoma. However, in vitro cytotoxicity tests revealed the occurrence of shared antigens between the RPC-5 and MOPC-315 tumor cells. It is concluded that the ineffectiveness of alkylating-drug treatment toward the RPC-5 tumor is not due to the inability of this tumor to induce a specific antitumor immune response, and that cross-antigenic relationship as revealed by in vitro cytotoxicity tests does not necessarily reflect cross-protection between various plasmacytomas.

THF-gamma 2, a synthetic thymic hormone, increases effectiveness of combined chemotherapy and immunotherapy against RPC-5 murine plasmacytoma.

The effect of a synthetic thymic hormone, THF-gamma 2, on the anti-tumor activity of spleen cells was studied in mice immunized against the RPC-5 tumor. Following two courses of the THF-gamma 2 treatment, the mean RPC-5 specific cytotoxic response of immune spleen cells was significantly increased when compared to normal cells (P less than 0.001) and to untreated immune spleen cells (P less than 0.04). In addition, THF-gamma 2 treatment improved the competence of immune spleen cells in adoptive immunotherapy (AIT) when performed in combination with chemotherapy by melphalan. Recipients of spleen cells from THF-gamma 2 treated mice showed a 35% increase in survival when compared to AIT with immune cells alone. The results suggest that THF-gamma 2 treatment of donors for AIT might be applicable to cancer therapy in humans.

Synergy between low-dose chemotherapy and immunotherapy in mouse L1210 leukemia.

A high dose of 4 mg/kg of daunorubicin (DAU) given in combination with immunostimulation by the P40 immunomodulatory fraction of Corynebacterium granulosum and glutaraldehyde (GA)-treated tumor cells coupled with tetanus toxoid (P40 + GA-L1210-Tet) was more effective than DAU alone for treatment of L1210 mouse leukemia. A combination of low doses of DAU (0.0625-0.25 mg/kg) and P40 + GA-L1210-Tet was more effective than either P40 + GA-L1210-Tet or DAU alone. The cured mice were resistant to challenge with a high tumorigenic dose of L1210 tumor cells. A combination of various doses of mitomycin (1-4 mg/kg) with P40 + GA-L1210-Tet was more effective than mitomycin alone and was at least as effective as P40 + GA-L1210-Tet alone. Administration of DAU (0.5-4 mg/kg) to noninoculated C57BL/6 X DBA/2 mice resulted in increase of stimulation in vitro of collected spleen cells by mitogens.

Simultaneous administration of diphtheria-tetanus-pertussis-polio and hepatitis B vaccines in a simplified immunization program: immune response to diphtheria toxoid, tetanus toxoid, pertussis, and hepatitis B surface antigen.

We studied the interactions of hepatitis B vaccine with other vaccines used in the World Health Organization expanded programs of immunization. Three groups of Senegalese children were vaccinated with hepatitis B vaccine (HB) alone, diphtheria-tetanus-pertussis (DTP)-polio vaccine alone, or a combination of hepatitis B vaccine and DTP-polio vaccines simultaneously. The immune responses to HBsAg, tetanus toxoid, diphtheria toxoid, and pertussis were measured after one and two vaccinations at 6-month intervals. The immune responses to the combination of HB vaccine and DTP-polio vaccines were similar to the immune responses observed after administration of each vaccine alone. In addition, no adverse reactions were noted. These experimental trials also demonstrated that with a DTP-polio vaccine containing 30Lf of tetanus and diphtheria toxoids, two doses given at 6-month intervals are sufficient to provide a satisfactory immune response. In the case of pertussis and HB vaccines; however, a third dose is necessary.

Combined tetanus-rabies vaccination.

Studies in mice have shown that Calcium Phosphate adsorbed tetanus toxoid (IPADT) can be used as a vehicle for freezedried rabies vaccine. Trials were undertaken in human volunteers and patients receiving a post-exposure treatment using the same vaccines to evaluate tolerance and antibody response to both vaccines.

Preparation and use of calcium phosphate adsorbed vaccines.

Simple or combined D,T,P and inactivated polio vaccines adsorbed onto calcium phosphate are prepared according to two procedures. Antigens can be dialysed in a sodium phosphate solution and quickly mixed with an equal volume of an equimolar solution of calcium chloride, the pH is adjusted to 6.8-7. Toxoids added to the phosphate solution are in this way entrapped within the 3-dimensional network during the formation of the precipitate. Antigens can also be added to a calcium phosphate gel suspension prepared in advance. Results of animal experiments and of field trials using either combined vaccines or simultaneous immunization with diphtheria, tetanus, pertussis, meningococcal and several viral vaccines: polio, rabies, hepatitis-B, etc., will be presented. Several programs have been studied in developing countries, mainly with the aim at simplifying vaccination campaigns. The efficiency of a two-dose regimen with DT vaccine has been ascertained, this has also been applied to pregnant women. Adsorbed tetanus toxoid was successfully used as a diluent for freeze-dried measles, meningococcal polysaccharide and rabies vaccines. Levels of circulating antibodies and potency of vaccines have been measured by new in vivo and in vitro methods. The choice of detoxified purified toxins is desirable for the preparation of vaccines in order to prevent the occurrence of adverse reactions. Local and generalized reactions have been studied. Adverse Arthus-type reactions have been encountered and related with the presence of high levels of circulating tetanus antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Simultaneous administration of diphtheria/tetanus/pertussis/polio vaccine and hepatitis B vaccine in a simplified immunization programme.

In most developing countries hepatitis B virus is endemic and prevention has to be carried out early in life and on a mass scale. In these regions, simultaneous administration of multiple antigens is normal practice. We have therefore, investigated the interaction of hepatitis B vaccine with DTP-Polio vaccine. Studies include the immune response post one and two injections to diphtheria and tetanus toxoid, pertussis and hepatitis B surface antigen. The vaccines were given simultaneously or not at a 6 months interval. The immune response to HBsAg vaccine and DTP-Polio vaccine injected simultaneously was equal to the immune response observed after administration of vaccines alone. Moreover, no adverse reactions were noted. This trial also demonstrated that immunization with two doses of DTP-Polio vaccine, containing 30 Lf of diphtheria and tetanus toxoids, at a 6 months interval is sufficient to obtain a very good immune response.

Calcium phosphate adjuvanted allergens.

Calcium phosphate has been used for many years as an adjuvant for vaccines. Results of field trials using calcium phosphate in several countries have been published and demonstrate high immunogenicity and the absence of untoward reactions. This paper presents preliminary results of studies on the use of calcium phosphate, instead of aluminum compounds, for preparing adsorbed allergens. These were performed with purified house dust, mite, and grass pollen extracts. Immunotherapy with house dust and mite extracts gave a high rate (70%) of satisfactory therapeutic results. Efficacy was compared with immunization carried out with fluid preparations under the same conditions. It was found that satisfactory results were of the same order, but elicited a higher number of injections. Immunotherapy with calcium phosphate adjuvanted allergens was also equal, as with aluminium adjuvanted allergens, according to published data. Good tolerance was noted for adsorbed grass pollen extracts, but clinical evaluation of results can only be reported after treatments during at least three seasons.

Studies on culture conditions of Bordetella pertussis and relationship to immunogenicity of vaccines.

Conditions for growing Bordetella pertussis bacteria in fermentors were studied for the purpose of producing highly protective vaccines. Bacteria with high protective potency were obtained in fermentors when the inoculum consisted of a dense suspension of young B. pertussis cells grown on Bordet-Gengou agar plates. When the time of cultivation in fermentors exceeded 30 h, bacteria harvested showed lower protective potency activity and lower levels of histamine sensitizing factor (HSF) and leukocytosis promoting factor (LPF), indicating that the optimal time for collection is at the end of the logarithmic growth phase. In fermentors, the ATP concentration in the bacterial cells increased from the time of inoculation until the end of the logarithmic growth phase. At the late logarithmic growth phase, the amounts of ATP, HSF, LPF and protective antigen declined rapidly. ATP kinetics thus provides a way of predicting the immunogenicity of vaccines. These results may be applicable to the production of vaccines exhibiting high protective activity at low bacterial concentrations, and to the preparation of soluble vaccines resulting from high-yield extraction of protective antigens. A correlation between the potency and LPF activity of vaccines was established for use in the rapid screening of vaccines for protective activity.

Prevention and treatment of L1210 mouse leukemia by immunization with xenogenized tumor cells combined with immunostimulation by the P40 fraction of C. granulosum and chemotherapy.

The effect on L1210 leukemia in mice of immunostimulation, in combination or not with chemotherapy with either daunorubicin or mitomycin, was studied. Immunostimulation with the immunomodulator P40 isolated from C. granulosum, together with xenogenized syngeneic tumor cells (GA-L1210-Tet: L1210 tumor cells inactivated with glutaraldehyde and coupled with tetanus toxoid), on days -14 and -7 before and days 2 and 9 after tumor inoculation, resulted in significant increase of the mean survival time as compared to control group with or without chemotherapy. Administration of P40 or P40 + GA-L1210-Tet cells, before or before and after inoculation of L1210 cells partly inactivated in vitro with antineoplastic agents, leads to a marked prolongation of mean survival time and to inhibition of ascitic tumor growth in a high percentage of mice. About 50% of the mice treated with P40 + GA-L1210-Tet cells and surviving the first challenge were resistant to rechallenge, providing that P40 + cells were reinjected before the second challenge, whereas all mice treated with P40 alone and surviving the first inoculation, were susceptible to rechallenge. The major conclusion is that treatments of combining chemotherapy, active immunization and nonspecific immunostimulation in the applied sequence are more effective than single treatments for control of L1210 mouse leukemia.